Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells.
We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3-4 mg (15)N-labeled protein suitable for NMR experiments. The (1)H-(15)N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.
Ohki
Shinya
S
Japan Advanced Institute of Science and Technology, Center for Nano Materials and Technology, Nomi, Ishikawa, Japan. shinya-o@jaist.ac.jp
Dohi
Koji
K
Tamai
Atsushi
A
Takeuchi
Makoto
M
Mori
Masashi
M
D016960:Agrobacterium tumefaciens / Q000378:metabolism; D007611:Aprotinin / Q000096:biosynthesis / Q000737:chemistry; D002147:Calmodulin / Q000096:biosynthesis / Q000737:chemistry; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D005822:Genetic Vectors; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry; D014026:Nicotiana / Q000378:metabolism*; D014162:Transfection
D002147:Calmodulin; D004220:Disulfides; D011994:Recombinant Proteins; D007611:Aprotinin; D013762:Tetrahydrofolate Dehydrogenase; D010749:Phosphoprotein Phosphatases